Most cases of genital herpes are due to herpes simplex virus (HSV)-2. The rate of HSV-2 infections increased by 30% from 1988 to 1994. In addition to genital herpes, transmission of HSV-2 to neonates causes severe life-threatening infections. Recent studies suggest a link between genital herpes and increased rates of transmission of HIV and elevated levels of HIV in the blood. HIV causes a number of opportunistic infections and is associated with increased rates of several cancers including lymphomas. Therefore, an effective HSV-2 vaccine is needed. Several HSV-2 vaccines have tested in humans for prevention or reduction genital herpes disease. A vaccine containing a single viral protein (HSV-2 glycoprotein D) reduced the rate of HSV-2 disease in women who were not previously infected with HSV-1 in two randomized, controlled clinical trials. The HSV-2 glycoprotein D vaccine, however, showed no efficacy in women previously infected with HSV-1 or in men. We postulated that the limited efficacy of the HSV-2 glycoprotein D vaccine is likely due to inadequate induction of cellular immune responses. We have been evaluating a candidate HSV-2 vaccine deleted for two essential genes, termed HSV-2 dl5-29, which was developed by David Knipe at Harvard University. Previously, we compared the cellular immune response of HSV-2 dl5-29 to glycoprotein D in mice and found that HSV-2 dl5-29 resulted in significantly higher systemic HSV-2 specific lymphocytes than glycoprotein D, and more rapid accumulation of HSV-2 specific lymphocytes in trigeminal ganglia after challenge with wild-type virus. Guinea pigs vaccinated with HSV-2 dl5-29 had higher levels of virus neutralizing antibodies in the sera compared with animals inoculated with recombinant HSV-2 glycoprotein D. Since the cellular immune and neutralizing antibody responses to HSV-2 dl5-29 were superior to that seen with recombinant HSV-2 glycoprotein D, HSV-2 dl5-29 is an attractive candidate for early phase clinical trials in humans. Evaluation of these vaccines in clinical trials requires that one can quantify antibody to the vaccine components and determine if persons have become infected with naturally occurring virus after vaccination. In 2009 we developed highly quantitative, high throughput serological assays to quantify antibody levels to several HSV proteins. These luciferase immunoprecipitation system (LIPS) assays are performed by constructing fusion proteins in which portions of HSV proteins are fused to a luciferase protein and human plasma or sera is used to immunoprecipitate the proteins. Since luciferase emits light, the amount of immunoprecipitated fusion protein is measured in the quantity of light units in the precipitate with a luminometer. We tested plasma samples from subjects seropositive for HSV-1 and/or HSV-2 or seronegative for HSV-1 and HSV-2 that had previously been analyzed by standard Western blotting and the Focus Plexus immunoassay. Our LIPS test that measures anti-HSV-1 glycoprotein G antibody titers was 96% sensitive and 96% specific for detecting HSV-1 infection, compared with the Focus immunoassay, and was 92% sensitive and 96% specific, compared with Western blotting. Our LIPS tests that measures anti-HSV-2 glycoprotein G precisely matched those for Western blotting, with 100% sensitivity and 100% specificity, and showed robust antibody titers in all the HSV-2-infected samples that were over 1,000 times higher than those in HSV-2-negative or HSV-1-positive samples. Thus these LIPS assays could very accurately distinguish antibodies to HSV-2 from antibodies to HSV-1. We also developed a LIPS assay to detect antibodies to three other HSV-2 proteins, glycoprotein D, glycoprotein B, and UL29 (also known as ICP8). Antibodies to these proteins were detected in many of the HSV-1- and/or HSV-2-infected plasma samples and showed higher levels of reactivity in plasma from HSV-2-infected persons than plasma from HSV-1-infected persons. Accurately measuring the levels of antibodies to HSV glycoprotein D and glycoprotein B will be important to quantify the immune response to these proteins in vaccinees who receive these proteins. Accurately measuring the level of antibodies to HSV UL29 (ICP8) would help to determine if persons infected with the HSV-2 dl5-29 vaccine, which is deleted for UL29,have become infected with natural virus after vaccination, since vaccinees could make antibodies to all of the HSV proteins, except UL29 and UL5. Thus, profiling of antibody responses to a panel of HSV proteins may be useful for characterizing individual atnibody responses to infection and for monitoring responses to vaccines.